A distinctive feature of prion protein (PrP) biogenesis, which appears central to an understanding of prion disease pathophysiology, is the observation that nascent PrP is synthesized in the form of at least three distinctive conformers that also differ in transmembrane topology. A growing body of evidence suggests that one of these, termed (Ctm)PrP, triggers a final common pathway of apoptosis occurring in response to both genetic and infectious prion disease, while another conformer, termed (Sec)PrP, is anti-apoptotic and neuroprotective. Work over the last decade has revealed a number of means by which the mix of conformers synthesized from an initially homogeneous population of nascent PrP chains can be altered. In particular, the signal sequence, and a charged region termed the Stop Transfer Effector (STE) sequence immediately preceding the transmembrane region, have been demonstrated to be sequence determinants of PrP topology. More recent data suggests that: i) other regions besides the signal sequence and STE sequence may contribute to regulation of PrP transmembrane topology and conformation, ii) interaction of distinct domains within PrP are involved in the protein's topogenesis, and iii) mapping of protein-protein interactions during translocation may provide useful information for conformer manipulation. Here we propose studies designed to i) explore new topogenic sequences and their interactions in cis, and ii) identify the partner proteins with which nascent PrP is involved in trans. We will then use this information to better understand the mechanism by which individual PrP conformers are generated, and the relationship of those mechanisms to signaling in infectious scrapie. In the long run, this work may also make possible the development of novel approaches to treatment of prion disease through conformer manipulation.